A hallmark of vaccination- or natural infection-induced humoral immunity is its longevity. However, there are fundamental gaps in our ability to appropriately program B cell responses, and this has hampered the development of efficacious vaccines for several pathogens including Clostridium difficile-associated disease. Persistent antibody mediated protection against pathogens is conferred by memory B cells that can respond rapidly to new antigen exposure as well as long-lived plasma cells that reside in the bone marrow and secrete specific antibody. NKT cells provide help to B cells, driving memory B cell and plasma cell differentiation and survival and thus enhance protective antibody responses against toxins, bacteria, and viruses. However, the mechanisms by which NKT cells enhance humoral immunity are poorly understood. Our newest data show that distinct NKT functional subsets exert diverse effects on memory B cell and plasma cell differentiation. Our long-term goal is to systematically develop an understanding of the mechanisms by which NKT cells regulate long-lived humoral immunity and use that knowledge to drive the development of new vaccines. The overall objective of this application is to understand how distinct functional subsets of NKT cells in different organs exert diverse effects on the production of C. difficile toxin-specific antibody and differentiation of memory B cells and long-lived plasma cells. We will therefore test the hypothesis that specialized functional subsets of NKT cells in secondary lymphoid organs, liver, and bone marrow differentially regulate antibody production, memory B cell induction and persistence of long-lived plasma cells. To do this, we will pursue three specific aims: Specific Aim 1 we will identify the mechanism by which NKT cells induce B cell memory. Specific Aim 2 we will identify the mechanism by which NKT cells enhance survival of plasma cells. Specific Aim 3 we will identify functional subsets of NKT cells that enhance antibody production. The approach is innovative because it applies the new concept that different NKT functional subsets have distinct effects on different aspects of humoral immunity and because it employs novel tools to measure NKT-driven memory B cell responses. The proposed research is significant because it is expected to expand our understanding of how NKT cells govern the direction, magnitude, and duration of humoral immunity. In doing so, we will provide the basis for new and/or improved vaccines that harness NKT- mediated humoral immunity, and do so in a manner that appropriately directs the fate of responding B cells.